EP0599262A2 - Stabilized superoxide-dismutase (SOD) compositions - Google Patents

Stabilized superoxide-dismutase (SOD) compositions Download PDF

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Publication number
EP0599262A2
EP0599262A2 EP93118801A EP93118801A EP0599262A2 EP 0599262 A2 EP0599262 A2 EP 0599262A2 EP 93118801 A EP93118801 A EP 93118801A EP 93118801 A EP93118801 A EP 93118801A EP 0599262 A2 EP0599262 A2 EP 0599262A2
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Prior art keywords
cyclodextrin
superoxide dismutase
mannitol
hydroxypropyl
sod
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German (de)
French (fr)
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EP0599262A3 (en
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Reiner Dr. Meyer
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Savient Pharmaceuticals Inc
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Boehringer Ingelheim International GmbH
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)

Definitions

  • the invention relates to stabilized superoxide dismutase (SOD) compositions in which the components SOD, a cyclodextrin and additionally a mono- or disaccharide or a sugar alcohol derived from a monosaccharide are present in combination, pharmaceutical compositions which contain these components, the combined use of the individual components for the production of medicaments and methods for the production of such stabilized compositions.
  • SOD superoxide dismutase
  • Eukaryotic cells mainly contain two forms of superoxide dismutases (SOD), one of which is primarily in the cytosol (Cu / Zn-SOD) and the other is primarily in the mitochondria (Mn-SOD).
  • SOD superoxide dismutases
  • Cu / Zn-SOD cytosol
  • Mn-SOD mitochondria
  • Mn enzyme is located in the matrix, including the inner membrane, although the Mn-SOD was also detected in the cytosol of the liver cells (Mc Cord JM et al. , In: Superoxide and Superoxide Dismutases [AM Michelson, JM Mc Cord, I. Fridovich, ed. ] Academic Press, NY, 129-138, 1977).
  • Mn-SOD Fe-SOD
  • prokaryotes In addition to Mn-SOD, there is also Fe-SOD in prokaryotes. The latter has also been detected in algae and protozoa and in some plant species (Bridges, SM, Salin, ML, Plant Physiol. 68 , 275-278, 1981).
  • mitochondrial membranes can be regarded as one of the most important locations for O2 formation in animal cells, so it is not surprising that mitochondria have their own special SOD (Mn-SOD).
  • the structural gene of a prokaryotic Mn-SOD ( E. coli ) was cloned and the chromosomal sod A gene was localized (Touati, D., J Bact. 155 , 1078-1087, 1983).
  • the 699 bp nucleotide sequence of a mitochondrial yeast Mn-SOD was elucidated and the primary structure of both the precursor and the mature protein derived from it - with molecular weights of 26123 Da for the precursor and 23059 Da for the mature protein (Marres, CAM et al. , Eur. J Biochem. 147 , 153-161, 1985).
  • the production of SOD in particular using the methods of DNA recombination, is known from the prior art and is described in many different ways.
  • the human Cu / Zn SOD can be produced according to EP-A 0 138 111, EP-A 0 180 964, WO 85/01503 or WO 91/06634.
  • the human Mn-SOD can be obtained by the process described in EP-A 0282 899.
  • the preparation of the EC-SOD is disclosed in EP-A 0 236 385 or in EP-A 0 492 447.
  • SOD SOD
  • conventional methods for example by extraction from cells and / or tissues of animal or human origin and subsequent purification, for example by means of chromatographic separation, is also described in many different ways (for example EP-A 0 188 053, DE-OS 3124 228 ).
  • chemically modified derivatives of SOD or SOD analogs are known and e.g. in EP-A 0 292 321, EP-A 0 483 113 or WO 89/012677.
  • the SODs to be used according to the invention also include all derivatives and analogs of the SODs with the known biological or enzymatic activity spectrum.
  • the cyclodextrins to be used according to the invention are cyclic oligosaccharides or cyclic polymers whose ring systems consist of six, seven or eight ⁇ -1,4-linked glucose units and, depending on their number of monomers, as ⁇ -, ⁇ - or ⁇ -Cyclodextrins can be called.
  • the cyclodextrins are known to form inclusion complexes with various biomolecules, such as fatty acids or amino acids, or to include them until they are saturated. From the group of cyclodextrins, the ⁇ -cyclodextrins or chemically modified derivatives thereof have preferably been used to produce stable, aqueous protein solutions or to check their toxicity.
  • the sugars to be used according to the invention are the monosaccharides, preferably the aldohexoses, ketohexoses and their derivatives and optical isomers, and the disaccharides, for example D (+) - glucose, D (+) - mannose, D (+) - galactose, D (-) - fructose, D (+) - sorbose, sucrose, lactose, maltose.
  • the monosaccharides preferably the aldohexoses, ketohexoses and their derivatives and optical isomers
  • disaccharides for example D (+) - glucose, D (+) - mannose, D (+) - galactose, D (-) - fructose, D (+) - sorbose, sucrose, lactose, maltose.
  • Suitable sugar alcohols are the monosaccharides reduced to the corresponding polyhydric alcohols, for example those which can be derived from the above-mentioned monosaccharides, with mannitol or D-mannitol (D-mannitol) being very particularly preferred.
  • EP-A 0 142 345 proposes stabilizing interferons by adding a protease inhibitor, or sugar alcohols have been described for achieving a stabilizing effect (EP-A 0 080 879).
  • human serum albumin EP-A 0 162 332
  • dextrans dextrins and other saccharides
  • EP-A 0 123 291 albumin with sugar or sugar alcohols
  • gelatin EP-A 0 091 258
  • certain buffer systems WO 88/09674
  • WO 90/03784 describes the use of ⁇ - and ⁇ -cyclodextrin derivatives, in particular hydroxypropyl- ⁇ -cyclodextrin (HPBCD), for stabilizing various biologically active proteins.
  • HPBCD hydroxypropyl- ⁇ -cyclodextrin
  • IL-2 interleukin-2
  • TNF tumor necrosis factor
  • m-CSF macrophage colony stimulating factor
  • HGH human growth hormone
  • TNF tumor necrosis factor
  • enzymatically active forms are oligomers from non-covalently associated subunits; in the case of Cu / Zn-SOD a dimer (Hartz & Deutsch, J Biol. Chem. 247, 7043-50 (1972)), in the case of Mn-SOD a tetramer (McCord JM et al. , Superoxide and Superoxide Dismutases , Academic Press, London (1977), pp. 129-38). Stabilizing additives are said to prevent the formation of insoluble high-molecular aggregates, but not to prevent the aggregation into the enzymatically active oligomer.
  • compositions of superoxide dismutase according to the invention are expediently prepared as freeze-dried preparations. After resolvation, such lyophilisates provide clear solutions with high enzymatic activity.
  • the solutions intended for freeze-drying can contain superoxide dismutase in concentrations of 0.1-60 mg / ml. Concentrations of ⁇ 5 mg / ml are preferred, particularly preferred of ⁇ 10 mg / ml. These solutions preferably contain hydroxypropyl- ⁇ -cyclodextrin in a proportion of 0.5-20% (weight / volume), very preferably 1-11%, in particular 1-6%, and mannitol in a proportion of 0.5-10%, very preferably 1- 5% (weight / volume).
  • the invention further relates to processes for the preparation of the formulations according to the invention.
  • the formulations according to the invention can be used for the treatment of diseases, for example inflammation, pulmonary fibrosis, bronchial pulmonary dysplasia, hyperoxia of the lungs, acute respiratory diseases (eg adult respiratory distress syndrome (ARDS), infantile respiratory distress syndrome (IRDS)), fibrosis after radiation exposure, Damage caused by reperfusion after ischemia, or to prolong the period of survival of isolated organs ex vivo.
  • diseases for example inflammation, pulmonary fibrosis, bronchial pulmonary dysplasia, hyperoxia of the lungs, acute respiratory diseases (eg adult respiratory distress syndrome (ARDS), infantile respiratory distress syndrome (IRDS)), fibrosis after radiation exposure, Damage caused by reperfusion after ischemia, or to prolong the period of survival of isolated organs ex vivo.
  • ARDS adult respiratory distress syndrome
  • IRDS infantile respiratory distress syndrome
  • a solution with additives was prepared in a buffer solution such that after the Mn-SOD-containing solution had been mixed with the buffer solution containing the additives, the desired final concentrations of all substances were set.
  • the formulation thus obtained was filled into ampoules at 1.0 ml. These filled ampoules were lyophilized (freezing to -40 ° C, holding for 2 hours at -40 ° C, main drying at -30 ° C and 0.02 mbar until the product temperature equals the shelf temperature, Post-drying at 0 ° C for 4 hours and at + 20 ° C for 2 hours; Freeze dryer Edwards / Kniese, Lyofex 0.4).
  • the ampoules were closed with stoppers and stored in the refrigerator, at room temperature and at 40 ° C. in the dark. The entire manufacturing process was carried out under sterile conditions. After certain time intervals (maximum 1/2 year) the lyophilisates were redissolved with water and analyzed. The nature of the lyocakes, the resolvate behavior, the resolvate, the specific Mn-SOD activity (McCord et Fridovich, J. Biol. Chem. 244 , 6049 (1969)) and any degradation products and aggregates were routinely analyzed using polyacrylamide gel electrophoresis (Laemmli, Nature 227: 680-5 (1970)).
  • formulations contained in the table which were prepared with a Mn-SOD concentration of 10 mg / ml, formulations were prepared from solutions which contained 0.1, 1.0 and 20 mg / ml Mn-SOD.

Abstract

Eine stabilisierte Superoxid-Dismutase (SOD)-Zusammensetzung enthält als Zusätze Cyclodextrin, vorzugsweise Hydroxypropyl-β-cyclodextrin, und entweder einen Zucker oder einen Zuckeralkohol, vorzugsweise Mannitol. Gefriergetrocknete Formulierungen mit der erfindungsgemäßen Zusammensetzung ergeben bei Resolvatation klare Lösungen mit hoher enzymatischer Aktivität.A stabilized superoxide dismutase (SOD) composition contains, as additives, cyclodextrin, preferably hydroxypropyl-β-cyclodextrin, and either a sugar or a sugar alcohol, preferably mannitol. Freeze-dried formulations with the composition according to the invention result in clear solutions with high enzymatic activity on resolvation.

Description

Die Erfindung betrifft stabilisierte Superoxid-Dismutase (SOD)-Zusammensetzungen, in denen die Bestandteile SOD, ein Cyclodextrin und zusätzlich ein Mono- oder Disaccharid oder ein von einem Monosaccharid abgeleiteter Zuckeralkohol in Kombination vorliegen, pharmazeutische Zusammensetzungen, die diese Komponenten enthalten, die kombinierte Verwendung der Einzelkomponenten zur Herstellung von Arzneimitteln sowie Verfahren zur Herstellung derartiger stabilisierter Zusammensetzungen.The invention relates to stabilized superoxide dismutase (SOD) compositions in which the components SOD, a cyclodextrin and additionally a mono- or disaccharide or a sugar alcohol derived from a monosaccharide are present in combination, pharmaceutical compositions which contain these components, the combined use of the individual components for the production of medicaments and methods for the production of such stabilized compositions.

Als Folge verschiedener biochemischer Prozesse in biologischen Systemen (z.B. Redoxprozesse in der Atmungskette, Oxydationen im Cytoplasma) werden bekannterweise laufend O₂.-Radikale gebildet, die hochcytotoxisch sind und zu Gewebezerstörungen führen können. Bei pathologischen Situationen, z.B. im Verlauf rheumatisch bedingter Erkrankungen, werden Degradationen von Collagen und Synovialflüssigkeit durch solche Radikale diskutiert (Pasquier, C. et al., Inflammation 8, 27-32, 1984).As a result of various biochemical processes in biological systems (eg redox processes in the respiratory chain, oxidations in the cytoplasm), it is known that O₂ radicals are formed continuously, which are highly cytotoxic and can lead to tissue destruction. In pathological situations, for example in the course of rheumatic diseases, degradations of collagen and synovial fluid by such radicals are discussed (Pasquier, C. et al. , Inflammation 8 , 27-32, 1984).

Eukaryotische Zellen enthalten hauptsächlich zwei Formen von Superoxiddismutasen (SOD), von denen die eine vornehmlich im Cytosol (Cu/Zn-SOD) und die andere vornehmlich in den Mitochondrien (Mn-SOD) vorliegt. In Lebermitochondrien wurde gefunden, daß das Mn-Enzym in der Matrix einschließlich der inneren Membran lokalisiert ist, obwohl die Mn-SOD auch im Cytosol der Leberzellen nachgewiesen wurde (Mc Cord J.M. et al., in: Superoxide und Superoxide Dismutases [A.M. Michelson, J.M. Mc Cord, I. Fridovich, Hrsg..] Academic Press, N.Y., 129-138, 1977).Eukaryotic cells mainly contain two forms of superoxide dismutases (SOD), one of which is primarily in the cytosol (Cu / Zn-SOD) and the other is primarily in the mitochondria (Mn-SOD). In liver mitochondria, it was found that the Mn enzyme is located in the matrix, including the inner membrane, although the Mn-SOD was also detected in the cytosol of the liver cells (Mc Cord JM et al. , In: Superoxide and Superoxide Dismutases [AM Michelson, JM Mc Cord, I. Fridovich, ed. ] Academic Press, NY, 129-138, 1977).

In Prokaryoten existiert neben einer Mn-SOD noch eine Fe-SOD. Letztere wurde auch in Algen und Protozoen sowie in einigen Pflanzenspezies nachgewiesen (Bridges, S.M., Salin, M.L., Plant Physiol. 68, 275-278, 1981).In addition to Mn-SOD, there is also Fe-SOD in prokaryotes. The latter has also been detected in algae and protozoa and in some plant species (Bridges, SM, Salin, ML, Plant Physiol. 68 , 275-278, 1981).

Diese hochaktiven Enzyme katalysieren die Disproportionierung O₂.⁻ + O₂.⁻ + 2H⁺ → H₂O₂ + O₂ und verhindern durch diese Dismutation der Superoxidradikale deren Anreicherung und somit deren zellschädigende Wirkung. Neben dem endoplasmatischen Retikulum der Leber können die mitochondrialen Membranen als einer der wichtigsten Orte der O₂-Entstehung in tierischen Zellen angesehen werden, sodaß es nicht verwundert, daß Mitochondrien ihre eigene, spezielle SOD (Mn-SOD) zur Verfügung haben.These highly active enzymes catalyze the disproportionation O₂.⁻ + O₂.⁻ + 2H⁺ → H₂O₂ + O₂ and prevent the superoxide radicals from accumulating and thus damaging the cells by this dismutation. In addition to the endoplasmic reticulum of the liver, the mitochondrial membranes can be regarded as one of the most important locations for O₂ formation in animal cells, so it is not surprising that mitochondria have their own special SOD (Mn-SOD).

Das Strukturgen einer prokaryotischen Mn-SOD (E. coli) wurde kloniert und das chromosomale sodA-Gen lokalisiert (Touati, D., J Bact. 155, 1078-1087, 1983).The structural gene of a prokaryotic Mn-SOD ( E. coli ) was cloned and the chromosomal sod A gene was localized (Touati, D., J Bact. 155 , 1078-1087, 1983).

Die 699 bp lange Nukleotidsequenz einer mitochondrialen Hefe Mn-SOD konnte aufgeklärt und die Primärstruktur sowohl des Precursors als auch des maturen Proteins davon abgeleitet werden - mit Molekulargewichten von 26123 Da für den Precursor und 23059 Da für das mature Protein (Marres, C.A.M. et al., Eur. J Biochem. 147, 153-161, 1985). Somit unterscheiden sich die Mn- und Cu/Zn-SOD (MG=14893, EP-A 138111) in ihren Molekulargewichten deutlich.The 699 bp nucleotide sequence of a mitochondrial yeast Mn-SOD was elucidated and the primary structure of both the precursor and the mature protein derived from it - with molecular weights of 26123 Da for the precursor and 23059 Da for the mature protein (Marres, CAM et al. , Eur. J Biochem. 147 , 153-161, 1985). The Mn and Cu / Zn SOD (MW = 14893, EP-A 138111) thus differ significantly in their molecular weights.

Die vollständige Aminosäuresequenz der Mn-SOD aus Menschenleber wurde von Barra, D. publiziert, wonach die hMn-SOD aus 196 Aminosäuren zusammengesetzt sein soll (Barra, D. et al., J. Biol. Chem. 259, 12595-12601, 1984). Die humane Cu/Zn-SOD aus Erythrocyten besteht demgegenüber aus 153 Aminosäuren (Jabusch, J.R., et al. Biochemistry 19, 2310-2316, 1980) und zeigt keine Sequenzhomologien zur hMn-SOD (Barra, D. et al., loc. cit.).The complete amino acid sequence of Mn-SOD from human liver was published by Barra, D., according to which the hMn-SOD was composed of 196 amino acids (Barra, D. et al. , J. Biol. Chem. 259 , 12595-12601, 1984). In contrast, the human Cu / Zn SOD from erythrocytes consists of 153 amino acids (Jabusch, JR, et al. Biochemistry 19 , 2310-2316, 1980) and shows no sequence homologies to hMn SOD (Barra, D. et al. , Loc. cit. ).

Die Herstellung der SOD, insbesondere über die Methoden der DNA-Rekombination, ist aus dem Stand der Technik bekannt und wird vielfältig beschrieben. Beispielsweise kann die humane Cu/Zn-SOD gemäß der EP-A 0 138 111, EP-A 0 180 964, WO 85/01503 oder WO 91/06634 hergestellt werden. Nach dem in der EP-A 0282 899 beschriebenen Verfahren ist die humane Mn-SOD erhältlich. In der EP-A 0 236 385 oder in der EP-A 0 492 447 wird die Herstellung der EC-SOD offenbart. Die Herstellung einer Fe-SOD wird gemäß der EP-A 218 480 ermöglicht und pflanzliche SODs werden in der EP-A 0 359 617, der EP-A 0 356 061 oder in der WO 90/01260 beschrieben. Eine Mn-SOD aus Serratia wird in der EP-A 0 210 761 offengelegt.The production of SOD, in particular using the methods of DNA recombination, is known from the prior art and is described in many different ways. For example, the human Cu / Zn SOD can be produced according to EP-A 0 138 111, EP-A 0 180 964, WO 85/01503 or WO 91/06634. The human Mn-SOD can be obtained by the process described in EP-A 0282 899. The preparation of the EC-SOD is disclosed in EP-A 0 236 385 or in EP-A 0 492 447. The production of an Fe-SOD is made possible according to EP-A 218 480 and vegetable SODs are described in EP-A 0 359 617, EP-A 0 356 061 or in WO 90/01260. An Mn-SOD from Serratia is disclosed in EP-A 0 210 761.

Die Herstellung und Gewinnung von SOD nach konventionellen Verfahren, beispielsweise durch Extraktion aus Zellen und/oder Geweben tierischer oder menschlicher Herkunft und anschließender Reinigung, beispielsweise über chromatographische Trennung, ist ebenfalls vielseitig beschrieben (z.B. EP-A 0 188 053, DE-OS 3124 228). Darüber hinaus sind chemisch modifizierte Derivate der SOD oder SOD-Analoga bekannt und z.B. in der EP-A 0 292 321 bzw. EP-A 0 483 113 oder WO 89/012677 offenbart.The production and recovery of SOD by conventional methods, for example by extraction from cells and / or tissues of animal or human origin and subsequent purification, for example by means of chromatographic separation, is also described in many different ways (for example EP-A 0 188 053, DE-OS 3124 228 ). In addition, chemically modified derivatives of SOD or SOD analogs are known and e.g. in EP-A 0 292 321, EP-A 0 483 113 or WO 89/012677.

Demzufolge umfassen die erfindungsgemäß zu verwendenden SODs auch alle Derivate und Analoga der SODs mit dem bekannten biologischen bzw. enzymatischen Aktivitätspektrum.Accordingly, the SODs to be used according to the invention also include all derivatives and analogs of the SODs with the known biological or enzymatic activity spectrum.

Bei den erfindungsgemäß zu verwendenden Cyclodextrinen handelt es sich um zyklische Oligosaccharide oder zyklische Polymere, deren Ringsysteme aus sechs, sieben oder acht α-1,4-verknüpften Glucose-Einheiten bestehen, und entsprechend ihrer Anzahl an Monomeren als α-, β- bzw. γ-Cyclodextrine bezeichnet werden. Von den Cyclodextrinen ist bekannt, daß sie mit verschiedenen Biomolekülen, wie z.B. Fettsäuren oder Aminosäuren, Inklusionskomplexe bilden, bzw. diese bis zur Sättigung einschließen können. Aus der Gruppe der Cyclodextrine sind vorzugsweise die β-Cyclodextrine bzw. chemisch modifizierte Derivate davon verwendet worden, um stabile, wässrige Proteinlösungen herzustellen bzw. um deren Toxizität zu überprüfen. Von der sehr großen Anzahl der bisher publizierten Literatur, die sich mit der Herstellung und Verwendung von Cyclodextrinen, insbesondere der ß-Cyclodextrine, befaßt, sollen nur beispielhaft die folgenden Publikationen genannt werden: Manning, M. et al., Pharm. Res. 6, 1903-1918, 1989; Yoshida, A. et al., Int. J. Pharm. 461, 217-222, 1988; Brewster, M. et al., Int. J. Pharm. 59, 231-243, 1980; Pitha, J. et al., Int. J. Pharm. 29, 73-82, 1986; Pitha, J. & Pitha J., J Pharm. Sci. 74, 987-990, 1985; Brewster, M. et al., J Parent. Sci. Technol. 43, 231-240, 1978. Insbesondere wird von Pitha J. & Pitha, J., loc. cit. (1986) die Herstellung von Hydroxypropyl-ß-cyclodextrin (HPBCD) beschrieben, und es wird berichtet, daß durch Verwendung von HPBCD die Wasserlöslichkeit der verschiedensten Wirkstoffe und biologischen Marklomoleküle verbessert wird. Hinsichtlich der Verwendung der Cyclodextrine auf pharmazeutischem Gebiet sei auf die Übersichtsartikel von Pitha, J. et al., in: Controlled Drug Delivery [Bruck, S.D., Hrsg.] Vol. I, CRC Press, Boca Raton, Florida, 125-148, 1983, verwiesen, oder auf Uekama, K., et al., in: CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 3 (1), 1-40, 1987; bzw. auf Uekama, K., in: Topics in Pharmaceutical Sciences 1987 [Breimer, D.D. and Speiser, P., Hrsg.] Elsevier Science Publishers B.V. (Biomedical Division), 181-194, 1987). Die Verwendung von insbesondere 2-Hydroxypropyl-β-cyclodextrin zur Solubilisierung und Stabilisierung von verschiedenen biologisch aktiven Proteinen wird von Brewster, M.E. et al., Pharm. Res. 8, 792-795, 1991, näher beschrieben.The cyclodextrins to be used according to the invention are cyclic oligosaccharides or cyclic polymers whose ring systems consist of six, seven or eight α-1,4-linked glucose units and, depending on their number of monomers, as α-, β- or γ-Cyclodextrins can be called. The cyclodextrins are known to form inclusion complexes with various biomolecules, such as fatty acids or amino acids, or to include them until they are saturated. From the group of cyclodextrins, the β-cyclodextrins or chemically modified derivatives thereof have preferably been used to produce stable, aqueous protein solutions or to check their toxicity. Of the very large number of previously published literature which deals with the production and use of cyclodextrins, in particular the β-cyclodextrins, the following publications should be mentioned only as examples: Manning, M. et al. , Pharm. Res. 6 , 1903-1918, 1989; Yoshida, A. et al. , Int. J. Pharm. 461 , 217-222, 1988; Brewster, M. et al. , Int. J. Pharm. 59 , 231-243, 1980; Pitha, J. et al. , Int. J. Pharm. 29 , 73-82, 1986; Pitha, J. & Pitha J., J Pharm. Sci. 74 , 987-990, 1985; Brewster, M. et al. , J Parent. Sci. Technol. 43 , 231-240, 1978. In particular, Pitha J. & Pitha, J., loc. cit. (1986) the preparation of hydroxypropyl-β-cyclodextrin (HPBCD), and it is reported that the use of HPBCD improves the water solubility of a wide variety of active ingredients and biological marrow molecules. Regarding the use of the cyclodextrins in the pharmaceutical field, reference is made to the review articles by Pitha, J. et al. , in: Controlled Drug Delivery [Bruck, SD, ed. ] Vol. I, CRC Press, Boca Raton, Florida, 125-148, 1983, or Uekama, K., et al. , in: CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 3 (1) , 1-40, 1987; and on Uekama, K., in: Topics in Pharmaceutical Sciences 1987 [Breimer, DD and Speiser, P., ed. ] Elsevier Science Publishers BV (Biomedical Division), 181-194, 1987). The use of in particular 2-hydroxypropyl-β-cyclodextrin for solubilizing and stabilizing various biologically active proteins is described by Brewster, ME et al. , Pharm. Res. 8 , 792-795, 1991.

Unter den erfindungsgemäß zu verwendenden Zuckern sind die Monosaccharide, vorzugsweise die Aldohexosen, Ketohexosen und ihre Derivate und optischen Isomere, und die Disaccharide zu verstehen, beispielsweise D(+)-Glucose, D(+)-Mannose, D(+)-Galactose, D(-)-Fructose, D(+)-Sorbose, Saccharose, Lactose, Maltose.The sugars to be used according to the invention are the monosaccharides, preferably the aldohexoses, ketohexoses and their derivatives and optical isomers, and the disaccharides, for example D (+) - glucose, D (+) - mannose, D (+) - galactose, D (-) - fructose, D (+) - sorbose, sucrose, lactose, maltose.

Als Zuckeralkohole kommen die zu den entsprechenden mehrwertigen Alkoholen reduzierten Monosaccharide in Frage, beispielsweise solche, die aus den oben erwähnten Monosacchariden abgeleitet werden können, wobei Mannitol bzw. D-Mannitol (D-Mannit) ganz besonders bevorzugt ist.Suitable sugar alcohols are the monosaccharides reduced to the corresponding polyhydric alcohols, for example those which can be derived from the above-mentioned monosaccharides, with mannitol or D-mannitol (D-mannitol) being very particularly preferred.

Viele Versuche wurden bisher unternommen, biologisch aktive Proteine durch Zusatz verschiedener Substanzen und Gemische zu stabilisieren.So far, many attempts have been made to stabilize biologically active proteins by adding various substances and mixtures.

Beispielsweise wird gemäß EP-A 0 142 345 vorgeschlagen, durch Zusatz eines Proteaseinhibitors Interferone zu stabilisieren, oder es wurden Zuckeralkohole für das Erreichen eines Stabilisierungseffektes beschrieben (EP-A 0 080 879). Neben diesen Stabilisatoren wurden auch Humanserumalbumin (EP-A 0 162 332), Dextrane, Dextrine und andere Saccharide (EP-A 0 123 291), Albumin mit Zucker oder Zuckeralkoholen (EP-A 0231 132), Gelatine (EP-A 0 091 258) oder bestimmte Puffersysteme (WO 88/09674) als stabilisierende Mittel bei biologisch aktiven Proteinen (Interferone, TNF, IL-2) offenbart.For example, EP-A 0 142 345 proposes stabilizing interferons by adding a protease inhibitor, or sugar alcohols have been described for achieving a stabilizing effect (EP-A 0 080 879). In addition to these stabilizers, human serum albumin (EP-A 0 162 332), dextrans, dextrins and other saccharides (EP-A 0 123 291), albumin with sugar or sugar alcohols (EP-A 0231 132), gelatin (EP-A 0 091 258) or certain buffer systems (WO 88/09674) as stabilizing agents for biologically active proteins (interferons, TNF, IL-2).

Die WO 90/03784 beschreibt die Verwendung von β- und γ-Cyclodextrin-Derivaten, insbesondere von Hydroxypropyl-β-cyclodextrin (HPBCD), zur Stabilisierung verschiedener biologisch aktiver Proteine. In dieser Patentanmeldung wird eine Stabilisierung oder Solubilisierung bzw. Aggregationsverhinderung mit insbesondere HPBCD explizit für Interleukin-2 (IL-2), Tumor-Nekrose-Faktor (TNF), Makrophagen-Kolonie-stimulierenden Faktor (m-CSF), Insulin und humanes Wachstumshormon (HGH) beschrieben.WO 90/03784 describes the use of β- and γ-cyclodextrin derivatives, in particular hydroxypropyl-β-cyclodextrin (HPBCD), for stabilizing various biologically active proteins. In this patent application a stabilization or solubilization or aggregation prevention with in particular HPBCD is explicitly for interleukin-2 (IL-2), tumor necrosis factor (TNF), macrophage colony stimulating factor (m-CSF), insulin and human growth hormone (HGH).

Der in der WO 90/03784 beschriebene Sachverhalt ist Gegenstand der Publikation von Brewster, M.E. et al., Pharm. Res. 8, 792-795, 1991.The situation described in WO 90/03784 is the subject of the publication by Brewster, ME et al. , Pharm. Res. 8 , 792-795, 1991.

Von Lee, M. und Fennema O.R., J Agric. Food Chem. 39, 17-21, 1991 ist bekannt, daß Cyclodextrine die Aggregationsbildung von β-Casein verhindern.By Lee, M. and Fennema OR, J Agric. Food Chem. 39 , 17-21, 1991 it is known that cyclodextrins prevent the formation of aggregation of β-casein.

Die Publikation von Hora M. S. et al., Pharm. Res. 9, 33-36, 1992, offenbart die Verhinderung der Bildung von löslichen Dimeren bei in lyophilisierter Form vorliegendem Tumor-Nekrose-Faktor (TNF), wenn vor der Lyophilisierung dem TNF Mannitol zusammen mit einer amorphen Komponente (Dextran, Saccharose, Trehalose oder 2-Hydroxypropyl-β -cyclodextrin) hinzugegeben wird.The publication by Hora MS et al. , Pharm. Res. 9 , 33-36, 1992, discloses the prevention of the formation of soluble dimers in the case of tumor necrosis factor (TNF) present in lyophilized form, if the TNF mannitol together with an amorphous component (dextran, Sucrose, trehalose or 2-hydroxypropyl-β-cyclodextrin) is added.

Wegen der potentiellen klinischen Bedeutung von SODs besteht ein Bedarf an stabilisierten Formulierungen dieser Proteine. Dies betrifft insbesondere lyophilisierte SOD-Präparate, die bei der Resolvatisierung klare Lösungen ergeben. Versuche, dies durch Zusätze wie Mannitol, Saccharose, Tween 20®, Tween 80® oder Serumalbumin zu erreichen, schlugen fehl.Because of the potential clinical importance of SODs, there is a need for stabilized formulations of these proteins. This applies in particular to lyophilized SOD preparations which give clear solutions when resolvated. Attempts to achieve this with additives such as mannitol, sucrose, Tween 20®, Tween 80® or serum albumin have failed.

Ein besonderes Problem der Superoxid-Dismutasen besteht darin, daß die enzymatisch aktiven Formen Oligomere aus nicht-kovalent assoziierten Untereinheiten sind; im Fall der Cu/Zn-SOD ein Dimer (Hartz & Deutsch, J Biol. Chem. 247, 7043-50 (1972)), im Fall der Mn-SOD ein Tetramer (McCord J.M. et al., Superoxide and Superoxide Dismutases, Academic Press, London (1977), S. 129-38). Stabilisierende Zusätze sollen zwar die Bildung unlöslicher hochmolekularer Aggregate, nicht jedoch die Aggregation zum enzymatisch aktiven Oligomer verhindern.A particular problem with superoxide dismutases is that the enzymatically active forms are oligomers from non-covalently associated subunits; in the case of Cu / Zn-SOD a dimer (Hartz & Deutsch, J Biol. Chem. 247, 7043-50 (1972)), in the case of Mn-SOD a tetramer (McCord JM et al. , Superoxide and Superoxide Dismutases , Academic Press, London (1977), pp. 129-38). Stabilizing additives are said to prevent the formation of insoluble high-molecular aggregates, but not to prevent the aggregation into the enzymatically active oligomer.

Demgegenüber stand kein Verfahren zur Verfügung, ein aus mehreren nicht-kovalent aggregierten Untereinheiten bestehendes Enzym wie Superoxid-Dismutase so zu formulieren, daß die Bildung unlöslicher Aggregate vermieden wird, ohne die Assoziation der Untereinheiten und damit die enzymatische Aktivität zu beeinträchtigen.In contrast, no method was available to formulate an enzyme consisting of several non-covalently aggregated subunits such as superoxide dismutase in such a way that the formation of insoluble aggregates is avoided without impairing the association of the subunits and thus the enzymatic activity.

Ein besonderes Problem stellte die Behandlung hochkonzentrierter Lösungen (10-60 mg/mi SOD) dar.The treatment of highly concentrated solutions (10-60 mg / ml SOD) was a particular problem.

Überraschenderweise wurde gefunden, daß der kombinierte Zusatz von HPBCD und einem oder mehreren Mono- oder Disacchariden bzw. von Monosacchariden abgeleiteten Zuckeralkoholen vor der Lyophilisation von SOD-Lösungen die Bildung unlöslicher hochmolekularer Aggregate verhindern konnte, nicht jedoch die erwünschte Aggregation zum enzymatisch aktiven Oligomer. Als besonders geeignet erwies sich die Kombination von HPBCD mit Mannitol.Surprisingly, it was found that the combined addition of HPBCD and one or more mono- or disaccharides or sugar alcohols derived from monosaccharides before the lyophilization of SOD solutions could prevent the formation of insoluble high-molecular aggregates, but not the desired aggregation to form the enzymatically active oligomer. The combination of HPBCD with mannitol has proven to be particularly suitable.

Die erfindungsgemäßen Formulierungen von Superoxid-Dismutase werden zweckmäßig als gefriergetrocknete Präparate hergestellt. Solche Lyophilisate liefern nach Resolvatisierung klare Lösungen mit hoher enzymatischer Aktivität.The formulations of superoxide dismutase according to the invention are expediently prepared as freeze-dried preparations. After resolvation, such lyophilisates provide clear solutions with high enzymatic activity.

Die zur Gefriertrocknung bestimmten Lösungen können Superoxid-Dismutase in Konzentrationen von 0.1-60 mg/mi enthalten. Bevorzugt sind Konzentrationen von ≧ 5 mg/ml, besonders bevorzugt von ≧ 10 mg/ml. Vorzugsweise enthalten diese Lösungen Hydroxypropyl-β-cyclodextrin in einem Anteil von 0.5-20 % (Gewicht/Volumen) ganz bevorzugt 1-11%, insbesondere 1-6 %, und Mannitol in einem Anteil von 0.5-10 %, ganz bevorzugt 1-5 % (Gewicht/Volumen).The solutions intended for freeze-drying can contain superoxide dismutase in concentrations of 0.1-60 mg / ml. Concentrations of ≧ 5 mg / ml are preferred, particularly preferred of ≧ 10 mg / ml. These solutions preferably contain hydroxypropyl-β-cyclodextrin in a proportion of 0.5-20% (weight / volume), very preferably 1-11%, in particular 1-6%, and mannitol in a proportion of 0.5-10%, very preferably 1- 5% (weight / volume).

Gegenstand der Erfindung sind ferner Verfahren zur Herstellung der erfindungsgemäßen Formulierungen.The invention further relates to processes for the preparation of the formulations according to the invention.

Die erfindungsgemäßen Formulierungen können zur Behandlung von Krankheiten verwendet werden, zum Beispiel Entzündungen, Lungenfibrose, bronchiale pulmonäre Dysplasie, Hyperoxie der Lunge, akute Atemwegserkrankungen (Z. B. adult respiratory distress syndrome (ARDS), infantile respiratory distress syndrome (IRDS)), Fibrose nach Strahlenexposition, durch Reperfusion nach Ischämie entstandene Schäden, oder zur Verlängerung der Überiebenszeit isolierter Organe ex vivo. Entsprechend sind pharmazeutische Zusammensetzungen, die die erfindungsgemäßen Zusammensetzungen sowie pharmazeutisch geeignete Träger- und/oder Hilfsstoffe enthalten, ebenfalls Gegenstand der Erfindung. Gegenstand der Erfindung ist ferner die kombinierte Verwendung der genannten Einzelkomponenten zur Herstellung von Arzneimitteln.The formulations according to the invention can be used for the treatment of diseases, for example inflammation, pulmonary fibrosis, bronchial pulmonary dysplasia, hyperoxia of the lungs, acute respiratory diseases (eg adult respiratory distress syndrome (ARDS), infantile respiratory distress syndrome (IRDS)), fibrosis after radiation exposure, Damage caused by reperfusion after ischemia, or to prolong the period of survival of isolated organs ex vivo. Accordingly, pharmaceutical compositions which contain the compositions according to the invention and pharmaceutically suitable carriers and / or auxiliaries are likewise the subject of the invention. The invention further relates to the combined use of the individual components mentioned for the production of medicaments.

Das folgende Beispiel erläutert die Erfindung.The following example illustrates the invention.

Beispielexample Experimentelles Verfahren zur Herstellung von Lyophilisaten und ResolvatenExperimental procedure for the production of lyophilisates and resolvates

Mn-SOD wurde gemäß EP-A 282 899 hergestellt. Hydroxypropyl-β-cyclodextrin ist bei ICN Biomedicals GmbH, Meckenheim, FRG, und D-Mannitol bei Serva Feinbiochemica GmbH & Co., Heidelberg, FRG, erhältlich.Mn-SOD was produced according to EP-A 282 899. Hydroxypropyl-β-cyclodextrin is available from ICN Biomedicals GmbH, Meckenheim, FRG, and D-Mannitol from Serva Feinbiochemica GmbH & Co., Heidelberg, FRG.

Das SOD-haltige Konzentrat (20 - 40 mg/ml Mn-SOD, 150 mM Natriumchlorid, 10 mM Natriumphosphat, pH 7.2) wurde gegen den Puffer dialysiert, der in der entsprechenden Formulierung verwendet werden sollte (siehe unten). Nach jeweils 2 Stunden wurde die Pufferlösung 3-malig gewechselt. Die Ausschlußgrenze des Dialyseschlauches war für Molekulargewichte ≧ 10000 Da.The SOD-containing concentrate (20-40 mg / ml Mn-SOD, 150 mM sodium chloride, 10 mM sodium phosphate, pH 7.2) was dialyzed against the buffer that should be used in the corresponding formulation (see below). After every 2 hours, the buffer solution was changed 3 times. The exclusion limit of the dialysis tube was Molekular 10,000 Da for molecular weights.

Die Dialyse war aber nicht notwendig, wenn die entsprechende Formulierung 10 mM Natriumphosphat und Natriumchlorid im Bereich von 75 - 150 mM enthalten sollte, da die Mn-SOD-Konzentration im Konzentrat hoch genug war, um nach Vermischen mit der Zusatzstoff-Lösung die gewünschte Endkonzentration der Zusatzstoffe erhalten zu können.However, dialysis was not necessary if the corresponding formulation should contain 10 mM sodium phosphate and sodium chloride in the range of 75-150 mM, since the Mn-SOD concentration in the concentrate was high enough to achieve the desired final concentration after mixing with the additive solution of the additives.

Eine Lösung mit Zusätzen wurde in einer Pufferlösung so angesetzt, daß nach dem Vermischen der Mn-SOD-haltigen Lösung mit der die Zusätze enthaltenden Pufferlösung die gewünschten Endkonzentrationen aller Substanzen eingestellt wurden. Die so erhaltene Formulierung wurde zu 1.0 ml in Ampullen abgefüllt. Diese abgefüllten Ampullen wurden lyophilisiert (Einfrieren auf -40°C, 2 Stunden halten bei -40°C, Haupttrocknung bei -30°C und 0.02 mbar bis Produkttemperatur gleich der Stellflächentemperatur, Nachtrocknung bei 0°C 4 Stunden und bei +20°C 2 Stunden; Gefriertrocknungsgerät Edwards/Kniese, Lyofex 0.4).A solution with additives was prepared in a buffer solution such that after the Mn-SOD-containing solution had been mixed with the buffer solution containing the additives, the desired final concentrations of all substances were set. The formulation thus obtained was filled into ampoules at 1.0 ml. These filled ampoules were lyophilized (freezing to -40 ° C, holding for 2 hours at -40 ° C, main drying at -30 ° C and 0.02 mbar until the product temperature equals the shelf temperature, Post-drying at 0 ° C for 4 hours and at + 20 ° C for 2 hours; Freeze dryer Edwards / Kniese, Lyofex 0.4).

Nach der Lyophilisation wurden die Ampullen mit Stopfen verschlossen und im Kühlschrank, bei Raumtemperatur und bei 40°C im Dunkeln gelagert. Der gesamte Herstellungsvorgang erfolgte unter sterilen Bedingungen. Nach bestimmten Zeitintervallen (maximal 1/2 Jahr) wurden die Lyophilisate mit Wasser wieder aufgelöst und analysiert. Routinemäßig analysiert wurden die Beschaffenheit der Lyokuchen, das Resolvatverhalten, das Resolvat, die spezifische Mn-SOD-Aktivität (McCord et Fridovich, J. Biol. Chem. 244, 6049 (1969)) sowie etwaige Abbauprodukte und Aggregate mittels Polyacrylamidgelelektrophorese (Laemmli, Nature 227, 680-5 (1970)).After lyophilization, the ampoules were closed with stoppers and stored in the refrigerator, at room temperature and at 40 ° C. in the dark. The entire manufacturing process was carried out under sterile conditions. After certain time intervals (maximum 1/2 year) the lyophilisates were redissolved with water and analyzed. The nature of the lyocakes, the resolvate behavior, the resolvate, the specific Mn-SOD activity (McCord et Fridovich, J. Biol. Chem. 244 , 6049 (1969)) and any degradation products and aggregates were routinely analyzed using polyacrylamide gel electrophoresis (Laemmli, Nature 227: 680-5 (1970)).

In der beigefügten Tabelle sind eine Reihe von Formulierungen zusammengefaßt, die nach diesem Muster hergestellt wurden. Die getesteten Zusatzstoffe waren: Natriumphosphatpuffer (Na-Phosphat), Natriumphosphat-Natriumcitrat-puffer (Phos-Citrat), Mannitol, Natriumchlorid (NaCl), Tween 80®, Cystein, Saccharose (Sucrose) und Hydroxypropyl-βcyclodextrin (HPBCD). Nach 1-2 Wochen Lagerzeit konnte bei den Formulierungen, die nicht die Kombination aus HPBCD und Mannitol enthielten, schon die ersten Trübungen bei 21° und 40°C Lagerung festgestellt werden. Die Formulierungen, die die Kombination aus HPBCD und Mannitol enthielten, zeigten auch noch bei 40°C nach 7 Monaten Lagerung keine trüben Resolvate.The attached table summarizes a number of formulations which were produced according to this pattern. The additives tested were: sodium phosphate buffer (Na phosphate), sodium phosphate sodium citrate buffer (Phos citrate), mannitol, sodium chloride (NaCl), Tween 80®, cysteine, sucrose (sucrose) and hydroxypropyl-βcyclodextrin (HPBCD). After 1-2 weeks of storage, the first turbidities at 21 ° and 40 ° C storage could already be determined for the formulations which did not contain the combination of HPBCD and mannitol. The formulations, which contained the combination of HPBCD and mannitol, showed no cloudy resolvates even at 40 ° C. after 7 months of storage.

Neben den in der Tabelle enthaltenen Formulierungen, die mit einer Mn-SOD-Konzentration von 10 mg/ml angesetzt wurden, wurden Formulierungen aus Lösungen hergestellt, die 0.1, 1.0 und 20 mg/ml Mn-SOD enthielten.In addition to the formulations contained in the table, which were prepared with a Mn-SOD concentration of 10 mg / ml, formulations were prepared from solutions which contained 0.1, 1.0 and 20 mg / ml Mn-SOD.

In den letzten 3 Spalten sind die Auswertungen der Beobachtungen aufgeführt, die den deutlichsten Unterschied zwischen den Formulierungen zeigten: Trübung der Resolvate nach Lagerung der Lyophilisate bei verschiedenen Temperaturen (5°C, 21°C, 40°C). Tabelle Resolvateigenschaften verschiedener MnSOD-Präparate (Lyophylisate) [MnSOD] (mg/ml) [Mannitol]¹ (%) [NaCl] (mM) [HPBCD] (%) Puffer Trübung² 5°C 21°C 40°C 10 5 Phos-Citrat 10 nM pH 7.2 0 0 ++ 10 2 6 Phos-Citrat 10 mM pH 7.2 0 0 0 10 1 11 Phos-Citrat 10 mM pH 7.2 0 0 0 10 61 6 Phos-Citrat 10 mM pH 7.2 0 + ++ 10 34 11 Phos-Citrat 10mM pH 7.2 0 0 ++ 10 2 6 Na-Phosphat 10 mM pH 7.2 0 0 0 10 61 6 Na-Phosphat 10 mM pH 7.2 0 ++ +++ 10 4.5 1 Na-Phosphat 10 mM pH 7.2 0 0 0 10 150 Na-Phosphat 10 mM pH 7.2 +++ 10 5 Na-Phosphat 10 mM pH 7.2 +++ +++ +++ 10 Sucrose (0.3 %) 8.6 Na-Phosphat 0.5 mM pH 7.2 + ¹ oder das angegebene Additiv ² Trübung: Die bei 3 Temperaturen (5°c, 21°C, 40°C) gelagerten Trockenampullen wurden nach der Resolvatisation mit dem Auge bemessen, wobei in 4 Stufen das Ausmaß der Trübung eingeteilt werden konnte: 0: klare Lösung; +: wenig Partikel und/oder sehr gering trüb; ++: viele Partikel und/oder gut sichtbar trüb; +++: stark trüb oder flockig In the last 3 columns, the evaluations of the observations are shown, which showed the clearest difference between the formulations: turbidity of the resolvates after storage of the lyophilisates at different temperatures (5 ° C, 21 ° C, 40 ° C). table Resolvate properties of various MnSOD preparations (lyophilisates) [MnSOD] (mg / ml) [Mannitol] 1 (%) [NaCl] (mM) [HPBCD] (%) buffer Turbidity² 5 ° C 21 ° C 40 ° C 10th 5 Phos citrate 10 nM pH 7.2 0 0 ++ 10th 2nd 6 Phos citrate 10 mM pH 7.2 0 0 0 10th 1 11 Phos citrate 10 mM pH 7.2 0 0 0 10th 61 6 Phos citrate 10 mM pH 7.2 0 + ++ 10th 34 11 Phos citrate 10mM pH 7.2 0 0 ++ 10th 2nd 6 Na phosphate 10 mM pH 7.2 0 0 0 10th 61 6 Na phosphate 10 mM pH 7.2 0 ++ +++ 10th 4.5 1 Na phosphate 10 mM pH 7.2 0 0 0 10th 150 Na phosphate 10 mM pH 7.2 +++ 10th 5 Na phosphate 10 mM pH 7.2 +++ +++ +++ 10th Sucrose (0.3%) 8.6 Na phosphate 0.5 mM pH 7.2 + ¹ or the specified additive ² Turbidity : The dry ampoules stored at 3 temperatures (5 ° C, 21 ° C, 40 ° C) were measured with the eye after the resolvation, the degree of turbidity being divided into 4 stages: 0: clear solution; +: few particles and / or very slightly cloudy; ++: many particles and / or visibly cloudy; +++: very cloudy or flaky

Claims (24)

Lösliche Zusammensetzung enthaltend Superoxid-Dismutase, Cyclodextrin und eine Substanz, die ausgewählt ist aus der Gruppe der Zucker und der Zuckeralkohole.Soluble composition containing superoxide dismutase, cyclodextrin and a substance selected from the group of sugars and sugar alcohols. Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, daß die Superoxid-Dismutase eine Mangan-Superoxid-Dismutase ist.Composition according to Claim 1, characterized in that the superoxide dismutase is a manganese superoxide dismutase. Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, daß das Cyclodextrin Hydroxypropyl-β-Cyclodextrin ist.Composition according to Claim 1, characterized in that the cyclodextrin is hydroxypropyl-β-cyclodextrin. Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, daß der Zuckeralkohol Mannitol ist.Composition according to claim 1, characterized in that the sugar alcohol is mannitol. Verfahren zur Herstellung einer löslichen Zusammensetzung enthaltend Superoxid-Dismutase und Cyclodextrin, dadurch gekennzeichnet, daß zusätzlich mindestens ein Mono- oder Disaccharid oder ein von einem Monosaccharid abgeleiteter Zuckeralkohol verwendet wird.Process for the preparation of a soluble composition containing superoxide dismutase and cyclodextrin, characterized in that additionally at least one mono- or disaccharide or a sugar alcohol derived from a monosaccharide is used. Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß der Zuckeralkohol Mannitol ist.A method according to claim 5, characterized in that the sugar alcohol is mannitol. Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß eine Lösung, enthaltend eine Superoxid-Dismutase, ein Cyclodextrin sowie zusätzlich mindestens ein Mono- oder Disaccharid oder einen von einem Monosaccharid abgeleiteten Zuckeralkohol, lyophilisiert wird.A method according to claim 5, characterized in that a solution containing a superoxide dismutase, a cyclodextrin and additionally at least one mono- or disaccharide or a sugar alcohol derived from a monosaccharide is lyophilized. Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß der Gehalt der Lösung an Superoxid-Dismutase 0.1-60 mg/ml beträgt.A method according to claim 7, characterized in that the content of superoxide dismutase in the solution is 0.1-60 mg / ml. Verfahren nach Ansprüchen 7 oder 8, dadurch gekennzeichnet, daß die Superoxid-Dismutase eine Mangan-Superoxid-Dismutase ist.Process according to claims 7 or 8, characterized in that the superoxide dismutase is a manganese superoxide dismutase. Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß das Cyclodextrin Hydroxypropyl-β-Cyclodextrin ist.Process according to claim 7, characterized in that the cyclodextrin is hydroxypropyl-β-cyclodextrin. Verfahren nach Anspruch 10, dadurch gekennzeichnet, daß der Gehalt der Lösung an Hydroxypropyl-β-cyclodextrin 0.5-20% (Gewicht/Volumen) beträgt.Process according to Claim 10, characterized in that the hydroxypropyl-β-cyclodextrin content of the solution is 0.5-20% (weight / volume). Verfahren nach Anspruch 10, dadurch gekennzeichnet, daß der Gehalt der Lösung an Hydroxypropyl-β-cyclodextrin 1-11% (Gewicht/Volumen) beträgt.A method according to claim 10, characterized in that the hydroxypropyl-β-cyclodextrin content of the solution is 1-11% (weight / volume). Verfahren nach Anspruch 10, dadurch gekennzeichnet, daß der Gehalt der Lösung an Hydroxypropyl-β-cyclodextrin 1-6% (Gewicht/Volumen) beträgt.Process according to Claim 10, characterized in that the hydroxypropyl-β-cyclodextrin content of the solution is 1-6% (weight / volume). Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß der Zuckeralkohol Mannitol ist.A method according to claim 7, characterized in that the sugar alcohol is mannitol. Verfahren nach Anspruch 14, dadurch gekennzeichnet, daß der Gehalt der Lösung an Mannitol 0.5-10% (Gewicht/Volumen) beträgt.A method according to claim 14, characterized in that the mannitol content of the solution is 0.5-10% (weight / volume). Verfahren nach Anspruch 14, dadurch gekennzeichnet, daß der Gehalt der Lösung an Mannitol 1-5% (Gewicht/Volumen) beträgt.A method according to claim 14, characterized in that the mannitol content of the solution is 1-5% (weight / volume). Pharmazeutische Zusammensetzung, enthaltend Superoxid-Dismutase, Cyclodextrin, eine Substanz, die ausgewählt ist aus der Gruppe der Zucker oder Zuckeralkohole sowie pharmazeutisch geeignete Träger- und/oder Hilfsstoffe.Pharmaceutical composition containing superoxide dismutase, cyclodextrin, a substance which is selected from the group of sugars or sugar alcohols and pharmaceutically suitable carriers and / or auxiliaries. Pharmazeutische Zusammensetzung nach Anspruch 17, dadurch gekennzeichnet, daß die Superoxid-Dismutase eine Mangan-Superoxid-Dismutase ist.Pharmaceutical composition according to claim 17, characterized in that the superoxide dismutase is a manganese superoxide dismutase. Pharmazeutische Zusammnesetzung nach Anspruch 17, dadurch gekennzeichnet, daß das Cyclodextrin Hydroxypropyl-β-cyclodextrin ist.Pharmaceutical composition according to claim 17, characterized in that the cyclodextrin is hydroxypropyl-β-cyclodextrin. Pharmazeutische Zusammensetzung, dadurch gekennzeichnet, daß der Zuckeralkohol Mannitol ist.Pharmaceutical composition, characterized in that the sugar alcohol is mannitol. Pharmazeutische Zusammensetzung nach einem der Ansprüche 17 bis 20 zur Behandlung von Entzündungen, Lungenfibrose, bronchialer pulmonärer Dysplasie, Hyperoxie der Lunge, akuten Atemwegserkrankungen wie adult respiratory distress syndrome (ARDS) oder infantile respiratory distress syndrome (IRDS), Fibrose nach Strahlenexposition, durch Reperfusion nach Ischämie entstandenen Schäden, oder zur Verlängerung der Überlebenszeit isolierter Organe ex vivo. Pharmaceutical composition according to one of claims 17 to 20 for the treatment of inflammation, pulmonary fibrosis, bronchial pulmonary dysplasia, hyperoxia of the lungs, acute respiratory diseases such as adult respiratory distress syndrome (ARDS) or infantile respiratory distress syndrome (IRDS), fibrosis after radiation exposure, by reperfusion Damage caused by ischemia, or to prolong the survival of isolated organs e vivo. Verwendung der Zusammensetzung nach Ansprüchen 1 bis 4 zur Herstellung eines Arzneimittels für die Behandlung von Erkrankungen oder zur Prophylaxe bei Säugetieren und beim Menschen.Use of the composition according to claims 1 to 4 for the manufacture of a medicament for the treatment of diseases or for prophylaxis in mammals and in humans. Verwendung von Superoxid-Dismutase, Hydroxypropyl-β-cyclodextrin und Mannitol zur Herstellung eines Arzneimittels für die Behandlung von Erkrankunegn oder zur Prophylaxe bei Säugetieren und beim Menschen.Use of superoxide dismutase, hydroxypropyl-β-cyclodextrin and mannitol for the manufacture of a medicament for the treatment of diseases or for the prophylaxis in mammals and humans. Verwendung nach Anspruch 22 oder 23, dadurch gekennzeichnet, daß das Arzneimittel für die Behandlung von Entzündungen, Lungenfibrose, bronchialer pulmonärer Dysplasie, Hyperoxie der Lunge, akuten Atemwegserkrankungen wie adult respiratory distress syndrome (ARDS) oder infantile respiratory distress syndrome (IRDS), Fibrose nach Strahlenexposition, durch Repertusion nach Ischämie entstandenen Schäden, oder zur Verlängerung der Überlebenszeit isolierter Organe ex vivo ist.Use according to claim 22 or 23, characterized in that the medicament for the treatment of inflammation, pulmonary fibrosis, bronchial pulmonary dysplasia, hyperoxia of the lungs, acute respiratory diseases such as adult respiratory distress syndrome (ARDS) or infantile respiratory distress syndrome (IRDS), fibrosis after Radiation exposure, damage caused by repertusion after ischemia, or to prolong the survival of isolated organs ex vivo .
EP19930118801 1992-11-27 1993-11-23 Stabilized superoxide-dismutase (SOD) compositions. Withdrawn EP0599262A3 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3091069A1 (en) * 2015-05-06 2016-11-09 Fitoplancton Marino S.L. Method for obtaining a biomass of a microalga of the species tetraselmis chuii enriched in superoxide dismutase (sod)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2719479B1 (en) * 1994-05-04 1996-07-26 Sanofi Elf Stable lyophilized formulation comprising a protein: assay kit.
US5876992A (en) * 1996-07-03 1999-03-02 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US6132712A (en) * 1996-12-11 2000-10-17 A. Glenn Braswell Superoxide dismutase stabilized with fragments of casein and pharmaceutical compositions incorporating same
US20040132653A1 (en) * 1997-01-30 2004-07-08 Biopharm Gmbh Lyophilized composition of bone morphogenetic factor human MP52
KR100548107B1 (en) * 1997-01-30 2006-02-02 바이오팜 게젤샤프트 쭈어 바이오테히놀로지셴 엔트비클룽 폰 파르마카 엠베하 Freeze-Dried Composition of Bone Morphogenetic Protein Human MP52
US20030022243A1 (en) * 2001-06-20 2003-01-30 Les Kondejewski Protein aggregation assays and uses thereof
DE10223783A1 (en) * 2002-05-29 2003-12-11 Boehringer Ingelheim Pharma New formulation for parenteral administration of a Na-channel blocker
KR100676502B1 (en) 2003-10-31 2007-02-02 김태윤 EC SOD and Cell transducing EC SOD and use thereof
US8318803B2 (en) * 2003-10-31 2012-11-27 Tae-Yoon Kim EC SOD and use thereof
KR101010055B1 (en) * 2008-06-16 2011-01-21 한국전력공사 Disk flow meter and its control method
CN102869775A (en) 2009-09-30 2013-01-09 哈佛大学校长及研究员协会 Methods for modulation of autophagy through the modulation of autophagy-inhibiting gene products
PT2919801T (en) * 2012-10-26 2020-07-30 Lupin Atlantis Holdings Sa Stable pharmaceutical composition of tnfr:fc fusion protein

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0284105A2 (en) * 1987-03-27 1988-09-28 Bio-Technology General Corporation Human manganese superoxide dismutase and methods of treatment
EP0355348A2 (en) * 1988-07-12 1990-02-28 Nippon Kayaku Kabushiki Kaisha Superoxide dismutase composition
WO1990003784A1 (en) * 1988-10-05 1990-04-19 Cetus Corporation Cyclodextrin-peptide complexes

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5712993A (en) * 1980-06-23 1982-01-22 Fujirebio Inc Isolation of superoxide dismutase
IT1142524B (en) * 1981-05-27 1986-10-08 Serono Ist Farm Antiinflammatory compsns. contg. orgotein
EP0080879B1 (en) * 1981-11-28 1986-10-01 Sunstar Kabushiki Kaisha Pharmaceutical composition containing interferon in stable state
HU189251B (en) * 1982-04-07 1986-06-30 Asahi Kasei Kogyo Kk,Jp Process for stabilizing tumor necrosis factor
EP0123291A2 (en) * 1983-04-20 1984-10-31 Kyowa Hakko Kogyo Co., Ltd. Method for stabilizing interferon
DE3486452T2 (en) * 1983-10-03 1997-11-06 Chiron Corp Superoxide dismutase and its expression in microorganisms
JPS60155133A (en) * 1983-11-09 1985-08-15 シンビオテイツクス　コ−ポレイシヨン Anti-idio type vaccine using anti-idio type monoclonal antibody
JPS60228422A (en) * 1984-04-26 1985-11-13 Suntory Ltd Stabilized preparation of physiologically active substance
CA1340597C (en) * 1984-08-27 1999-06-22 Haim Aviv Expression vectors containing pl promoter, and engineered restriction site for convenient replacement of ribosomal binding site, plasmids containing the vectors, hosts containing the plasmids and related methods
JPS61111690A (en) * 1984-11-06 1986-05-29 Ube Ind Ltd Recombinant dna and its use
CA1232850A (en) * 1985-01-17 1988-02-16 Albert D. Friesen Purification of superoxide dismutase
ES8800982A1 (en) * 1985-07-05 1987-12-01 Takeda Chemical Industries Ltd Modified enzymes, production and use thereof.
DK402785D0 (en) * 1985-09-03 1985-09-03 Syn Tek Ab PROCEDURE FOR THE PREPARATION OF AN ENZYM
JPS6279778A (en) * 1985-10-04 1987-04-13 Suntory Ltd Novel superoxide dismutase and production thereof
IE59498B1 (en) * 1985-11-22 1994-03-09 Bio Technology General Corp Human manganese superoxide dismutase analog, plasmid for its expression and method of recovering it in enzymatically active form
US5246847A (en) * 1985-11-22 1993-09-21 Bio-Technology General Corp. Polypeptide analogs of human manganese superoxide dismutase and compositions and complexes thereof
US4933433A (en) * 1986-01-31 1990-06-12 E. I. Du Pont De Nemours And Company Recombinant interleukin-2 composition and process for making it
US5260204A (en) * 1987-03-14 1993-11-09 Boehringer Ingelheim International Gmbh Human manganese superoxide dismutase (hMn-SOD)
EP0292321B1 (en) * 1987-05-21 1994-08-31 Suntory Limited Acylated derivative of superoxide dismutase and composition containing same
US4895716A (en) * 1987-06-09 1990-01-23 Biogen, Inc. Stabilized formulations of gamma interferons
US5002935A (en) * 1987-12-30 1991-03-26 University Of Florida Improvements in redox systems for brain-targeted drug delivery
JPH01304882A (en) * 1988-06-03 1989-12-08 Ube Ind Ltd Stable storing method of superoxide dismutase of human cu, zn-type
WO1989012677A1 (en) * 1988-06-14 1989-12-28 Chiron Corporation Superoxide dismutase analogs having novel binding properties
US5538878A (en) * 1988-08-08 1996-07-23 Calgene, Inc. Superoxide dismutase expression in plants
EP0359617A3 (en) * 1988-09-02 1990-04-04 Plant Genetic Systems, N.V. Stress-tolerant plants
IL96136A (en) * 1989-11-07 2000-12-06 Bio Technology General Corp Use of hydrophobic chromatography in a method for purification of copper-zinc superoxide dismutase from cells containing same
JPH0568541A (en) * 1990-12-28 1993-03-23 Asahi Chem Ind Co Ltd Method for producing active extracellular superoxide dismutase
US5298410A (en) * 1993-02-25 1994-03-29 Sterling Winthrop Inc. Lyophilized formulation of polyethylene oxide modified proteins with increased shelf-life

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0284105A2 (en) * 1987-03-27 1988-09-28 Bio-Technology General Corporation Human manganese superoxide dismutase and methods of treatment
EP0355348A2 (en) * 1988-07-12 1990-02-28 Nippon Kayaku Kabushiki Kaisha Superoxide dismutase composition
WO1990003784A1 (en) * 1988-10-05 1990-04-19 Cetus Corporation Cyclodextrin-peptide complexes

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3091069A1 (en) * 2015-05-06 2016-11-09 Fitoplancton Marino S.L. Method for obtaining a biomass of a microalga of the species tetraselmis chuii enriched in superoxide dismutase (sod)
US11473065B2 (en) 2015-05-06 2022-10-18 Fitoplancton Marino, S.L. Method for obtaining a biomass of a microalga of the species Tetraselmis chuii enriched in superoxide dismutase (SOD)

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